Shah Niket, Kuntz Douglas A, Rose David R
Department of Medical Biophysics, University of Toronto, Toronto, ON, Canada.
Proc Natl Acad Sci U S A. 2008 Jul 15;105(28):9570-5. doi: 10.1073/pnas.0802206105. Epub 2008 Jul 3.
Golgi alpha-mannosidase II (GMII) is a key glycosyl hydrolase in the N-linked glycosylation pathway. It catalyzes the removal of two different mannosyl linkages of GlcNAcMan(5)GlcNAc(2), which is the committed step in complex N-glycan synthesis. Inhibition of this enzyme has shown promise in certain cancers in both laboratory and clinical settings. Here we present the high-resolution crystal structure of a nucleophile mutant of Drosophila melanogaster GMII (dGMII) bound to its natural oligosaccharide substrate and an oligosaccharide precursor as well as the structure of the unliganded mutant. These structures allow us to identify three sugar-binding subsites within the larger active site cleft. Our results allow for the formulation of the complete catalytic process of dGMII, which involves a specific order of bond cleavage, and a major substrate rearrangement in the active site. This process is likely conserved for all GMII enzymes-but not in the structurally related lysosomal mannosidase-and will form the basis for the design of specific inhibitors against GMII.
高尔基体α-甘露糖苷酶II（GMII）是N-连接糖基化途径中的一种关键糖基水解酶。它催化去除GlcNAcMan(5)GlcNAc(2)的两种不同甘露糖基连接，这是复杂N-聚糖合成中的关键步骤。在实验室和临床环境中，抑制这种酶已在某些癌症中显示出前景。在这里，我们展示了与天然寡糖底物和寡糖前体结合的黑腹果蝇GMII（dGMII）亲核突变体的高分辨率晶体结构以及未结合配体的突变体结构。这些结构使我们能够在较大的活性位点裂隙内识别出三个糖结合亚位点。我们的结果有助于阐明dGMII的完整催化过程，该过程涉及特定的键断裂顺序以及活性位点中的主要底物重排。这个过程可能在所有GMII酶中都是保守的——但在结构相关的溶酶体甘露糖苷酶中并非如此——并将为设计针对GMII的特异性抑制剂奠定基础。
